The gene PIG3 is induced by the tumor suppressor p53 but not by p53 mutants unable to induce apoptosis, suggesting its involvement in p53-mediated cell death. Here we show that p53 directly binds and activates the PIG3 promoter, but not through the previously described DNA element. Instead, p53 interacts with a pentanucleotide microsatellite sequence within the PIG3 promoter (TGYCC)n where Y=C or T. Despite its limited similarity to the p53-binding consensus, this sequence is necessary and sufficient for transcriptional activation of the PIG3 promoter by p53 and binds specifically to p53 in vitro and in vivo. In a population of 117 healthy donors from Germany, the microsatellite was found to be polymorphic, the number of pentanucleotide repeats being 10, 15, 16 or 17, and the frequency of alleles 5.1%, 62.0%, 21.4% and 11.5%, respectively. The number of repeats directly correlated with the extent of transcriptional activation by p53. This is the first time that a microsatellite has been shown to mediate the induction of a promoter through direct interaction with a transcription factor. Moreover, this sequence of PIG3 is the first p53-responsive element found to be polymorphic. Inheritance of this microsatellite may affect an individual's susceptibility to cancer.

The tumor suppressor p53 regulates transcription positively and negatively, depending on the target gene. Whereas p53 induces transcription through direct interaction with promoter DNA, the mechanism of p53-mediated transcriptional repression is less well understood. Early reports described the alleviation of p53-mediated repression by inhibitors of apoptosis, suggesting that negative regulation of transcription might occur only in conjunction with programmed cell death. More recently, it has been proposed that certain genes, such as survivin, are repressed by direct association of p53 with their promoters, followed by recruitment of a repressor complex. We show here that p53-mediated negative regulation of transcription could occur independently of apoptosis. In contrast, the amino-terminal transactivation domain of p53 was required for negative regulation of transcription. Similarly, the p53 homologue p73 diminished the expression of survivin and stathmin, depending on its transactivation domain. Mutation of the putative p53 binding site within the survivin promoter did not impair its repression. These observations raised the hypothesis that activation of an effector gene might be required for repression by p53. Strikingly, when the p53-inducible p21/CDKN1A gene was deleted, p53 no longer repressed any one among 11 genes that it downregulates otherwise. Most of these genes were also repressed by ectopic p21 in the absence of p53. Overexpressed c-Myc reduced the transcription of p21/ CDKN1A and impaired p53-mediated repression but did not abolish repression by ectopic p21. Taken together, these results strongly suggest that increased expression of p21/CDKN1A is necessary and sufficient for the negative regulation of gene expression by p53.p53 is a key regulator of cell growth and apoptosis. Its central role in tumor suppression becomes evident by the fact that the p53 gene is mutated in about 50% of human malignancies. p53 acts as a transcription factor, modulating the expression of growth and death regulators. As a result, cell proliferation is suppressed, and/or programmed cell death is induced (1). It is generally accepted that p53 activates a number of promoters through direct interaction with the promoter DNA and the subsequent recruitment of the basal transcription machinery, e.g. the TFIID complex and the p300/CPB histone acetyl transferases. A tetramer of p53 molecules is assembled through the carboxyl-terminal oligomerization domains. This allows the central domains to interact directly with a consensus DNA element. As a consequence, the amino-terminal transactivation domains interact with basal transcription factors,

The p53 tumor suppressor protein activates transcription and induces cell death. A close homologue of p53, termed p73, is expressed in transactivating (TA) forms that induce growth arrest and apoptosis much like p53. However, the p73 gene contains a second promoter, giving rise to the expression of p73DN, a species of p73 proteins that lack the N-terminal transactivation domain. We show here that the expression of p73DN is induced by p53 on the mRNA and protein level. The promoter that regulates p73DN expression in human cells was cloned and found to be activated by p53, as well as by p73TA, directly through a specific DNA element. The p73DN proteins, that are thereby expressed, bound to p53-responsive promoter DNA, competed with p53 for DNA binding, antagonized the activation of transcription by p53, and prevented p53-induced cell death. In addition, a transcriptional repressor domain was identified within the splicing variant p73DNa. The combination of p73DNa and mdm2 antagonized p53 more strongly than either p73Na or mdm2 alone. Blocking endogenous p73DN by a trans dominant fragment, or its removal by siRNA, increased the activity of a p53-responsive promoter in cells that contain a wild type p53 gene. Thus, the induction of p73DN expression by p53 establishes an autoregulatory feedback loop that keeps the trigger of cell death under tight control.

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The p53 tumor suppressor protein induces apoptosis through a mechanism that may involve the transcriptional activation of cellular genes, including the PIG3 gene. A p53 protein lacking the proline-rich region (p53D62-91) induces many p53-responsive genes but not PIG3. In parallel, this mutant induces growth arrest but not apoptosis. We show here that the replacement of the N-terminal (amino acids 1 ± 80) or C-terminal (amino acids 344 ± 393) domains of p53 with heterologous domains does not interfere with transcription from the PIG3 promoter, but these chimeras still require the proline-rich region for PIG3 activation. The p53-homolog p73b also activated the PIG3 promoter, but in contrast to p53, the proline-rich domain of p73b (residues 81 ± 113) was dispensable to induce the PIG3 promoter. Some tumor-derived p53-mutants, especially M246I, retained the ability to activate transcription of mdm2 but speci®cally failed to induce the PIG3 promoter, thus resembling p53D62-91. Further, p53D62-91 and p53M246I were defective for induction of apoptosis. Finally, p53D62-91 and p53M246I both showed reduced binding to the DNA of the PIG3 promoter and also to the DNA of the mdm2 and p21 promoters in vitro. Correspondingly, at low expression levels, p53D62-91 and p53M246I poorly activated the mdm2 promoter when compared to wild type p53. Our results suggest that the proline-rich domain of p53 a ects the ability of the central domain to bind DNA. Moreover, some tumor-derived mutations within the central DNA binding domain of p53 mimic the loss of the proline-rich domain.

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